1. Field of the Invention
This invention relates generally to compositions and methods for inhibiting angiogenesis. In particular, this invention relates to compositions comprising intraceptors, and methods of use thereof, to disrupt the intracellular expression and/or secretion of a vascular endothelial growth factors (VEGF) and/or vascular endothelial growth factor receptor (VEGFR). In certain embodiments, such intraceptors can be used to treat angiogenesis related conditions.
2. Background Art
Angiogenesis, the growth of new blood vessels, is a fundamental biological process which plays a central role in the pathogenesis of various conditions, and is a major contributor to mortality and morbidity in diseases, such as cancer, diabetic retinopathy, and macular degeneration (Folkman, 1990, JNCI 82: 4-6). Cancer is the second leading cause of death in the United States, claiming 553,251 lives in 2001 (National Vital Statistics Report. 2003). Diabetic retinopathy affects over 5.3 million Americans, and is the leading cause of new blindness among U.S. adults 20-74 years of age. Proliferative diabetic retinopathy (PDR) is a condition in which abnormal new blood vessels in the retina may rupture and bleed inside the eye, and is a principal cause of blindness in diabetics. The prevalence of PDR increases from 2% at diagnosis to 20% after 20 years of disease (Morbidity and Mortality Weekly Report, 1993, pp. 191-95). The estimated incidence of new PDR cases is about 65,000 per year. Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among those over 65 in the United States, Western Europe, and Japan and affects over 11 million persons in the US. More than 20% of the American population is older than 55 years of age and at risk for AMD. Each year, more than a million individuals suffer severe central vision loss due to AMD; these numbers will skyrocket with the aging population. Neovascular AMD is responsible for severe vision loss in 80-90% of these patients (Macular Photocoagulation Study Group. Arch Opthalmol. 1991; 109:1109-14).
Corneal neovascularization is a central feature in the pathogenesis of many blinding corneal disorders, and a major sight-threatening complication in corneal infections, chemical injury, and following keratoplasty, in which neovascularization adversely impacts corneal transplant survival (Epstein et al., 1987, Cornea. 6:250-57). Anti-angiogenic molecules have been shown to inhibit corneal neovascularization (Ambati et al, 2002, Arch Opthalmol. 120: 1063-68). Thermal laser and photodynamic therapy induces only temporary closure of new vessels (Primbs et al., 1998, 29: 832-38), without addressing the underlying biology of neovascularization.
Annually, approximately 45,000 corneal transplants are performed in the US. This is the highest number for any transplant, largely because of high success conferred by the immune privilege of the normally avascular cornea. Corneal neovascularization breaches this immune privilege, and is a major factor in rejection of corneal transplants, which occurs in about 10% of cases.
Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis in many models (Neufeld et al., 1999, FASEB J. 13: 9; Dvorak, 1999, Curr. Top Microbiol. Immunol. 237: 97; and Carmeliet & Collen, 1999, Curr. Top Microbiol. Immunol. 237: 133, etc.). VEGF promotes vascular endothelial cell migration, proliferation, inhibition of apoptosis, vasodilation, and increased vascular permeability. In several clinically relevant models of animal and human corneal neovascularization, angiogenesis is driven by increased secretion of VEGF-A (herein referred to as VEGF) (Amano et al., 1998, Invest Opthalmol Vis Sci. 18-22; Cursiefen et al., 2004, J. Clin Invest. 113: 1040-50; and Philipp et al., 2000, Invest Opthalmol Vis Sci. 41: 2514-22), and is also closely linked to infiltrating leukocytes (Amano et al., 1998, Invest Opthalmol Vis Sci. 18-22).
Three receptors constitute the VEGF receptor family, which includes VEGFR-1 (Flt or Flt-1), VEGFR-2 (KDR), and VEGFR-3 (Flt-4), all of which have tyrosine-kinase activity (Neufeld et al., 1999, FASEB J. 13:9). The cDNA and amino acid sequences of human Flt-1 are found at accession number gi:56385329. Several studies have shown VEGFR-2 (through activation of MAP kinase and P1-3K (phosphatidylinositol 3-kinase) is the signal transducer for VEGF-induced mitogenesis, chemotaxis, and cytoskeletal reorganization and thus the principal receptor involved in angiogenesis (Thakker et al., 1999, J. Biol. Chem. 274: 10002-7; Dimmeler et al., 2000, FEBS Lett 477: 258-62; Carmeliet & Collen, 1999, Curr. Top Microbol. Immunol. 237:97; Neufeld et al., 1999, FASEB J. 13:9; and Millauer et al., 1993, Cell 72: 835-46). VEGFR-3 is primarily involved in lymphangiogenesis (Cursiefen et al., 2004, Invest Opthalmol Vis Sci. 45: 2666-73; Cursiefen et al., 2004, J. Clin Invest. 113: 1040-50).
VEGF transcription is amplified in response to oncogenes, hypoxia, and other insults. Transcription factors for VEGF (HIF-1α and HIF-2α) are stabilized during hypoxia (Ahmed et al., 2000, Placenta 21 SA:S16-24; Wenger & Gassman, 1997, Biol. Chem. 378: 609). Sensitivity to hypoxia is a major difference between VEGF and other angiogenic factors (Arbiser et al., 1997, Proc. Natl. Acad. Sci. USA 94: 861; Okada et al., 1998, Proc. Natl. Acad. Sci. USA 95: 3609; and Petit et al., 1997, Am. J. Pathol. 151:1523). Elevated VEGF has been associated with a poor prognosis in cancer and with diabetic retinopathy (Ambati et al., 1997, Arch Opthalmol 115: 1161-66). Strategies to inhibit VEGF have included blocking antibodies, decoy receptors for VEGF, and anti-VEGF antibodies (Kim et al., 1993, Nature 362: 841-44; Yuan et al., 1996, Proc. Natl. Acad. Sci. USA 93: 14765-70; Lin et al., 1998, Cell Growth Differ. 9: 49-58; and Hasan & Jayson, 2001, Expert Opin Biol Ther. 1: 703-18). These strategies have generally reduced neovascularization by only 30-50% (Robinson et al., 1996, Proc. Natl. Acad. Sci. USA 93: 4851-56; Aiello et al., 1995, Proc. Natl. Acad. Sci. USA 92: 10457-61; Shen et al., 2002, Lab Invest 82: 167-82; and Honda et al., 2000, Gene Ther. 7: 978-75). These levels of neovascularization reduction are insufficient for the cornea, where angiogenesis should be minimized as much as possible for optimal visual clarity.
Further, in the course of normal VEGF signal transduction, membrane Flt heterodimerizes with VEGFR-2 upon VEGF binding (Autiero et al., 2003, Nat. Med.; and Kendall et al., 1996, Biochem Biophy Res Comm. 226:324-28). Physiologic Flt/VEGFR-2 heterodimers stimulate expression of the genes for the transcription factor Ets-1 and matrix metalloproteinase 1 (MMP-1), phosphorylation of focal adhesive kinase (FAK), vinculin assembly, and DNA synthesis (Kanno et al., 2000, Oncogene 19: 2138-46; Sato et al., 2000, Ann NY Acad. Sci. 902:201-7). Ets-1 induces expression of matrix metalloproteinase 1 (MMP-1), MMP-3, MMP-9, urokinase plasminogen activator, and β3 integrin b, all involved in matrix-neovessel interactions. MMP-1 facilitates digestion of extracellular matrix to facilitate vascular ingrowth, while FAK helps mediate adhesion among endothelial cells and extracellular matrix. These events are critical to endothelial cell migration and proliferation.
KDEL (SEQ ID NO:1) is the one letter sequence for the peptide retention signal having the amino acid sequence Lys-Asp-Glu-Leu (SEQ ID NO:1) which binds endoplasmic reticulum retention receptors, thus preventing secretion of ligands of proteins coupled to the sequence (Pelham, 1990, Trends Biochem Sci. 15: 483-6). This is also a highly specific retention sequence, as constructs using a KDEV (SEQ ID NO:2) sequence are not successful at retaining targets (Tang et al., 1992, J. Biol. Chem. 267: 7072-6). Although the mechanism of clearance or degradation of KDEL-sequestered (SEQ ID NO:1) proteins is not fully known, the ubiquitin-proteasome pathway is thought to be the principal route for clearance of intraceptor-retained proteins, as removal of KDEL (SEQ ID NO:1) from PDI, an ER chaperone has recently been described to release its target protein, procollagen 1, from ubiquitin-proteasome degradation (ko & Kay, 2004, Exp Cell Res. 295: 25-35).
Linkage of KDEL (SEQ ID NO:1) to chemokines (known as creation of “intrakines”) downregulates cognate receptors with significant roles in disease (Chen et al., 1997, Nat. Med. 3: 1110-6; Kreitman et al., 1995, Cancer Res. 55: 3357-63). Coupling stromal derived factor (SDF) with KDEL (SEQ ID NO:1) blocked cell surface expression of SDF's receptor, CXCR-4; similar efforts have been used to down-regulate cell surface expression of other receptors, including CCR-5 and Interleukin-4 receptor (Kreitman et al., 1995, Cancer Res. 55: 3357-63; Luis et al., 2003, Mol. Ther. 8: 475-84; and Steinberger et al., 2000, Proc Natl Acad. Sci. 97:805-10).
It has been reported that sequestered proteins are eventually degraded in the endoplasmic reticulum (Pelham, 1990, Trends Biochem Sci. 15: 483-6). The accumulation of sequestered proteins in the endoplasmic reticulum may lead to endoplasmic reticulum overload, triggering the unfolded protein response (UPR), which could cause apoptosis of endothelial cells, as the presence of unfolded proteins in endoplasmic reticulum (ER) leads to a stress response including release of pro-apoptotic factors such as CHOP and caspase-12 (Wang et al., 1998, EMBO J. 17: 5708-17; Yoshida et al., 2001, Cell 107: 881-91; Tirasophon et al., 1998, Genes Dev. 12: 812-24; Fomace et al., 1988, Proc Natl Acad. Sci. USA 85: 8800-4; Kaufman, 2002, J Clin Invest 110: 1389-98; and Schroder & Kaufman, 2005, Mutat Res. 569: 29-63). Although the effect of KDEL (SEQ ID NO:1)-mediated protein retention on these molecular responses is unknown, it has been reported that the KDEL (SEQ ID NO:1) receptor is involved in the ER stress response (Yamamoto et al., 2003, J. Biol. Chem. 278: 34525-32).
VEGF is an important target for inhibiting angiogenesis. Molecular interventions such as anti-VEGF aptamers or antibodies (e.g. Macugen; Eyetech) and ranibizumab (Lucentis; Genentech) are currently used or under investigation for AMD and PDR, but are based on extracellular blockade of VEGF. Intracellular approaches against VEGF could potentially ameliorate these conditions, as results for extracellular modalities have been mixed (Gragoudas et al., 2004, N Engl J Med 351: 2805-16), or add a new additional means to affectively reduce total VEGF function.
It is important to target VEDF intracellularly, as several cell types respond to their own VEGF production in an autocrine fashion. VEGF autocrine loops have also been demonstrated in endothelial cells (Honda et al., 2000, Gene Ther. 7: 978-75; Lee et al., 1999, Eur J Cancer 35: 1089-93), including in hypoxic HUVEC cells (Lee et al., 1999, Eur J Cancer 35: 1089-93; Liu & Ellis, 1998, Pathobiology 66: 247-52); further, VEGF can upregulate its own receptor VEGFR-2 (Shen et al., 1998, J. Biol. Chem. 273: 29979-85). Cancer cells producing VEGF and VEGFR-2 include prostate carcinoma, leukemia, pancreatic carcinoma, melanoma, Kaposi's sarcoma, and osteosarcoma (Lee et al, 1999, Eur J Cancer 35: 1089-93; Masood et al., 2001, Blood 98: 1904-13). Intracellular autocrine loops render cells resistant to modalities targeting VEGF extracellularly (Gerber et al., 2002, Nature 417: 954-58; Santos & Dias, 2004, Blood 103: 3883-9).
Intracellularly disrupting VEGF expression is potentially superior to extracellular blockade by antibodies or aptamers as intracellular gene silencing may sabotage intracellular autocrine loops that have been demonstrated for VEGF in cancer and endothelial cells (Lee et al., 1999, Eur J. Cancer. 35: 1089-93; Liu & Ellis, 1998, Pathobiology 66: 247-52; Casella et al., 2003, Blood 101: 1316-23; Gerber et al., 2002, Nature 417: 954-58; Straume & Akslen, 2001, Pathol. 159: 223-35). Intracellular disruption of VEGF signaling may represent a powerful addition to the anti-angiogenic arsenal, by sabotaging VEGF secretion and intracellular autocrine loops.
Alternative gene silencing approaches relying on RNAi, antisense oligonucleotides or ribozymes for disrupting VEGF expression, and approaches to sequester VEGF using PIGF-KDEL (SEQ ID NO:1) are previously described in the art. However, since placental growth factor (P1GF) can heterodimerize with VEGF or a complex of an anti-VEGF Fab fragment with KDEL (SEQ ID NO:1; Wheeler et al., 2003, FASEB J. 17: 1733-5), there is a need to develop a more effective approach for disrupting both VEGF and VEGFR-2 intracellularly for treating or preventing angiogenesis. Such approach can induce the unfolded protein response in cells that produce VEGF, resulting in selective ER stress. Furthermore, such approach is able to disrupt physiologic heterodimer formation of Flt/VEGFR-2, providing high specificity and affinity for the target molecule due to the use of a receptor as the therapeutic substance.